CN102703426A - Method for constructing a nucleic acid library, reagent and kit - Google Patents
Method for constructing a nucleic acid library, reagent and kit Download PDFInfo
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Abstract
The invention provides a method, reagent and a kit for establishing a nucleic acid library with a mark and uses an in vitro transposition method; the method comprises the following steps: firstly, breaking target DNA at random by transposase, and then, treating the target DNA by nuclease and DNA polymerase to generate a DNA library with a mark; according to the invention, any DNA segment which needs a mark is allowed to be connected, so that the generated DNA segment with the mark can be directly applied to different second generation of sequencing platforms which include but are not limited to those instruments manufactured by companies of Illumina, Life Technologies (ABI), Roche, HelicosBiotechnologies, Pacific Biosciences; similarly, the above-mentioned DNA segment can also be applied to sequencing instruments produced by other manufacturers.
Description
Technical field
The present invention provides method, reagent and test kit for setting up nucleic acid library.Transposase, nucleicacidase and ligase enzyme particularly in building the storehouse process, have been used.
Background technology
How effectively with efficiently, set up the library with the DNA and the RNA of various differing materials, this is a challenge to the check order researchist and the operator of (NGS) of the s-generation.Existing technology, comprise through physical means: supersonic method or atomization interrupt double-stranded DNA, and this needs more a large amount of parent materials.This process is very long, is difficult to carry out with high-throughout mode.The scientist of NEB develops recently and uses Fragmentase, in centrifuge tube, carries out enzyme reaction, and this interrupts with physics, and to compare be a good selection, but still a large amount of parent material of needs.EPICENTRE company has developed Nextera reagent, builds the storehouse process thereby the researchist can be accomplished in 2 hours, and as long as parent material is 50ng.Yet the sequencing primer that it needs and the sequencing primer of existing order-checking platform are incompatible, and this is to let many potential users bring down a bit.
Another challenge that s-generation order-checking Jian Ku field faces is, the development of order-checking platform is very fast, and the research and development of new sequenator only need the time in several years, and it is very fast to update.Third generation sequencing technologies also might be in the near future can throw down the gauntlet or replaces s-generation technology.The fast development of new technology and equipment, for those need carry out very little adjustment, just can with database technology be applied to new instrument and technology platform build the storehouse product brought the technology and the coml competitive power, also brought huge commercial opportunity for those enterprises.
The objective of the invention is to set up the preparation method in a library; Can be used for all new sequenators existing and the few change that is about to release, can be used for high-throughput, and be applicable to machine operation; Only need less parent material, can produce satisfied sequencing result.
Summary of the invention
The invention provides method, component and the test kit of random fracture and while labeled dna fragment.Particularly, the present invention adopts external swivel base, comes the labeling nucleic acid storehouse to use to be fit to order-checking of future generation with nucleicacidase and ligase enzyme.
In some instances, the inventive method is that random fracture and while labeled dna fragment provide method, and suitable dna fragmentation can be the genomic dna of purifying; Double-stranded cDNA; Double-stranded DNA product behind the PCR, or the large fragment DNA of rolling-circle replication generation, the source of these DNA can be the people; Animal, plant or mikrobe.
Comprising: a) transposase and transposon end sequence (Transposone End-TE) are mixed with double-stranded target dna, under the katalysis of transposase, double-stranded transposon inserts in the target dna sequence at random.Transposase can comprise: (Tn5, Tn3, Tn7, Tn8, Tn9, Tn10, Tn11, Tn2, Tn4, Tn6 and their various sudden change versions.In the transposon insertion process, a terminal strand of transposon is connected 5 ' of dna segment with covalent linkage and holds, and the terminal second chain of transposon does not link to each other with dna segment, but under annealing conditions, still exists with double chain form.The room of 9 bases is arranged in that the swivel base of second chain is terminal between holding with dna fragmentation 3 '.Just produced random fracture thus, and by the dna fragmentation of (TE) mark.These fragments from 5 ' to 3 ' start from double-stranded transposon end sequence, are the single stranded DNA sequence of 9 base pairs then, are double-stranded target dna fragment then, and the single stranded DNA sequence of 9bp and double-stranded transposon are terminal; B) from the dna segment of transposon end sequence (TE) mark, remove double-stranded transposon end sequence (TE), generate unlabelled dna fragmentation; C) dna marker that connection needs on unlabelled dna segment produces a new markd dna fragmentation.
In some instances, transposon can comprise: (Tn5), the transposon end sequence can comprise (TE).
In instances more of the present invention, the single stranded DNA restriction endonuclease is used to cut the strand zone of last 9 bases of DNA of 9 base transposon end sequences (TE) mark, and purpose is to remove double-stranded transposon end sequence.Many single stranded DNA restriction endonucleases can be removed the double-stranded transposon end sequence in DNA two ends with this with removing single stranded DNA, produce to have the flat terminal double-stranded DNA segment that is not labeled.In the other instance, mend flat 5 ' end with archaeal dna polymerase, add the A tail at 3 ' end.The polysaccharase that is applicable to this application not only is confined to archaeal dna polymerase and Taq archaeal dna polymerase, and large fragment DNA polysaccharase I (Klenow fragment) also can use.The Klenow fragment is a dna polymerase i of having removed 3 ' to 5 ' 5 prime excision enzyme activity.And mend flat 5 ' end with archaeal dna polymerase, and form on flat terminal (rather than A tail), then will be with the archaeal dna polymerase that 3 ' to 5 ' 5 prime excision enzyme activity is arranged, like the T7DNA polysaccharase, T4DNA polysaccharase, big segment dna polymerase i etc.
In some instances, the second chain that transposon is terminal is a free, and the discord target dna sequence is covalently bound.Under hot conditions, the dna fragmentation of transposon end sequence (TE) mark is separated, and target dna partly remains unchanged.After removing the terminal second chain of transposon, can use some specific single stranded DNA nucleicacidases to remove article one chain of remaining transposon and the single stranded DNA of 9 base pairs.
In some embodiments of the present invention, need be coupled with the label of particular requirement by the target dna of random fracture, the dna fragmentation that produces mark is to be applicable to the needs of downstream application.Dna ligase can couple together the DNA of mark and dna segment like the T4DNA ligase enzyme.According to the dna fragmentation of downstream application needs, can add required label: like the order-checking label, the barcode label, address tag, the restriction enzyme site mark is caught mark, certification mark, amplification label, or transcripting promoter sequence label.The use that also can link together of a plurality of marks.Two ends at unlabelled double chain DNA fragment can connect identical or different label.In some instances, required sequence label is provided by many two generations order-checkings company, like Illumina, and Life Technologies, Roche, Complete Genomics and Pacific Biosciences etc.
In certain embodiments, the invention provides a kind of random fracture that is used for, and form the test kit of markd dna fragmentation, comprising the transposon of purifying, transposon terminal (TE) and single stranded DNA nucleicacidase.This test kit can also comprise archaeal dna polymerase, for example, and dna polymerase i, large fragment DNA polysaccharase I (Klenow fragment) or Taq archaeal dna polymerase.In certain embodiments, this test kit also comprises dna ligase, for example, and the T4DNA ligase enzyme.In certain embodiments, this test kit can also comprise required label, can be one or more order-checking labels, the barcode label, and address tag, the restriction enzyme site mark is caught mark, certification mark, amplification label, or transcripting promoter sequence label.In certain embodiments, required sequence label is provided by many two generations order-checkings company, like Illumina, and Life Technologies, Roche, Complete Genomics and Pacific Biosciences etc.
The present invention is employed in external transposon system, and the target dna random fracture is to required segment size, terminal with the equating of single stranded DNA nucleicacidase, and comes labeled dna fragment at the required label of flat terminal connection of DNA.These fragments that are labeled can be used for various application, comprise the analysis of the macro genome DNA of patient's sample, icp gene group analysis, genome sequencing, SNP gene type, in situ hybridization.
The invention provides the dna fragmentation library that a kind of effective means produces the random fracture of mark, adapt to various order-checking platform.An advantage of the invention is that it allows to connect the dna fragmentation of any required mark, make the dna fragmentation of the mark that produces; Can be applied directly to different s-generation order-checking platforms, include but not limited to by Illumina Life Technologies (ABI); Roche; HelicosBiotechnologies, the instrument of these manufactured of Pacific Biosciences equally also is applicable to the sequenator of other manufacturers produce.Another advantage of the present invention is that method of the present invention can improve flux at an easy rate, and carries out automated operation by robot.
Technical term " transposon is terminal " or " transposon end sequence " are meant the double-stranded DNA of being made up of specific nucleotide sequence.It can form one at the external swivel base complex body that the swivel base function is arranged with transposase.This is to carrying out the swivel base reaction time to insert double-stranded target DNA be vital external.The transposon end is a double chain DNA sequence; Article one, chain (transferable chain) can be covalently bound with 5 ' end of target DNA; Second chain (non-transfer chain) directly is not connected with target DNA in the swivel base process, but is that annealed reaction is connected with the terminal article one chain (transferable chain) of swivel base.Specific transposon end sequence can discerned and use to each transposase in the swivel base reaction.
" transposase " is meant the enzyme that can form the function mixture with the swivel base end sequence, and the mixture of formation can be inserted in the target DNA by catalysis transposon end sequence.The provirus intergrase is also included within the transposase.
" swivel base reaction " or " external swivel base reaction " is meant that the transposase mixture can interrupt target DNA, is inserted in the transposon end sequence in the target DNA segment.When the two ends of same target DNA change two transposons over to, the dna segment between those two transposons is separated opening.Therefore the swivel base reaction can interrupt and carries out mark endways the purpose segment at random.The swivel base process can produce the single stranded DNA of 9 bases between target dna and transposon end sequence.
Description of drawings
Fig. 1: the DNA of the same race of identical original bulk is in the difference between molecular weight area after the different buffer solution system cleaved.A uses the interval damping fluid of lower molecular weight; B uses the interval damping fluid of HMW.
Fig. 2: new-generation sequencing technology DNA library construction result: 1,1kb Plus DNA Ladder (Life Technologies, 10787-018); 2, human gene group DNA behind the 50ng purifying; 3, and after the 50ng human gene group DNA adopts rolling-circle replication (illustra GenomiPhi V2 DNA amplification Kit, GE Health Care Life Sciences, 25-6600-30); 4,50ng PCR product (fragment that 4kb obtains from human gene group DNA's amplification with particular probe, Phusion Hot Start Flex 2X Master Mix, NEBM0536L).
Fig. 3: M, and 1kb Plus DNA Ladder (Life Technologies, 10787-018); 1, corn gene group DNA Fig. 4: M behind the 100ng purifying, 1kb Plus DNA Ladder (Life Technologies, 10787-018); 1, hammer vibrios DNA behind the 50ng purifying; 2, the hammer vibrios DNA that interrupts obtains double chain DNA fragment through PCR;
Embodiment
Embodiment 1, the structure in end-labelled DNA library is arranged
Utilize transposase and transposon end sequence (TE) that target DNA is interrupted at random, make it reach the clip size of our expectation.Usually we place target DNA and transposase and specific swivel base end sequence thereof the damping fluid of swivel base reaction to hatch.Add target DNA (for example human gene group DNA), transposase (EZ-Tn5 Transposase, EPICENTRE) and the amount of specific swivel base end sequence (0.01ng/ul-1ug/ul) according to different application and different.The composition of damping fluid and concentration (1 * GPSBuffer, 25mM Tris-HCl, 2mM DTT), temperature of hatching and time thereof also will decide according to the size of want cutting fragment.As shown in Figure 1: as in two kinds of different buffer solution systems, to be different between the molecular weight area that DNA is ruptured.Adding reaction terminating liquid (10% sucrose, 66mM EDTA, 20mM TRIS, 0.1%SDS, 0.9%Orange G, and 100g/ml Proteinase K), 10 minutes afterreactions of 50 ℃ of heating can stop.The size that can on 1% sepharose (V2111 of Promega company) electrophoresis, check post-rift dna fragmentation.
In case the swivel base reaction is accomplished, the dna fragmentation of transposon end sequence (TE) mark is that (Zymo Research Corporation, Irvine CA) carries out purifying to available ZymoDNAClean and Concentrator kit.
(S1nuclease, Promega) (exoVII, mixture Promega) is hatched at 37 ℃, up to the terminal equating of the dna fragmentation of strand with excision enzyme for the dna fragmentation of transposon end sequence (TE) mark that purifying is good and single stranded DNA restriction endonuclease.Come purifying not have the DNA of mark with ZymoDNA Clean and Concentrator kit.The end sequence of transposon so far is removed, and has produced unlabelled dna fragmentation.
In the unlabelled dna fragmentation of above generation, add the big fragment of dna polymerase i of no 5 prime excision enzyme activity, promptly the Klenow fragment was hatched 20 minutes at 37 ℃.Dna fragmentation through this processing can link to each other with oligonucleotide joint.
The dna fragmentation that does not have mark, the label that checks order certainly, the mixture of the T4DNA ligase enzyme that the order-checking label of barcode label and ligation are used was handled 5 minutes or was spent the night at 16 ℃.Add EDTA or stopped ligation in 20 minutes 72 ℃ of reactions.The dna fragmentation library that has label promptly can be used to downstream analysis.
Parent material with Tn5 at 50 ℃ of 5 minutes (Nextera DNA Sample Preparation Kit of LMW; EPICENTRE; GA09115); Then 37 degree with S1 nuclease (Promega M5761) is hatched 30 minutes. the dna fragmentation that adds a tail that obtains with Klenow from 3 ' to 5 ' circumscribed interrupting (NEB, 0212L); 20minutes.16 ℃ of 5 minutes use fast link test kit link order-checking street corner of 37 ℃ of hatchings. the tagged DNA that obtains carries out pcr amplification (Phusion Hot Start Flex 2X Master Mix according to the PCR operating process of standard with following system; NEB M0536L). (Zymo Research Corporation, Irvine CA) come purifying and enrichment to all samples with ZymoDNA Clean and Concentrator kit in the process of at every turn handling with enzyme; The PCR product that obtains is at 1% agarose gel electrophoresis and with SYBRgreen I Nucleic Acid dyeing (Life technologies; S7567), resulting result see the picture 2 that takes with Safe Imager Blue Light Transil luminator (Life technologies, G6600).
Embodiment 2
The genomic DNA fragment that adopts corn utilizes transposase and transposon end sequence (TE) that target DNA is interrupted at random as material, makes it reach the clip size of our expectation.The buffer system that material adopts is (1 * GPS Buffer, 25mM Tri s-HCl), under different incubation conditions (37 ℃; 20 minutes) handle sample, add reaction terminating liquid (10% sucrose, 66mM EDTA then; 20mM TRIS, 0.1%SDS, 0.9%Orange G; And 100g/ml Proteinase K), 10 minutes afterreactions of 50 ℃ of heating can stop.Can under 1% gel electrophoresis, observe cracked dna fragmentation size.
In case the swivel base reaction is accomplished, the dna fragmentation of transposon end sequence (TE) mark is that (Zymo Research Corporation, Irvine CA) carries out purifying to available ZymoDNAClean and Concentrator kit.
(S1nuclease Promega) is hatched at 37 ℃ with the mixture of excision enzyme, up to the terminal equating of the dna fragmentation of strand for the dna fragmentation of transposon end sequence (TE) mark that purifying is good and single stranded DNA restriction endonuclease.Come purifying not have the DNA of mark with ZymoDNA Clean and Concentrator kit.The end sequence of transposon so far is removed, and has produced unlabelled dna fragmentation.
In the unlabelled dna fragmentation of above generation, add the big fragment of dna polymerase i of no 5 prime excision enzyme activity, promptly the Klenow fragment was hatched 20 minutes at 37 ℃.Dna fragmentation through this processing can link to each other with oligonucleotide joint.
The dna fragmentation that does not have mark,, catch mark, the mixture of the T4DNA ligase enzyme that the order-checking label of certification mark and ligation are used was handled 5 minutes or was spent the night at 16 ℃.Add EDTA or stopped ligation in 20 minutes 72 ℃ of reactions.The dna fragmentation library that has label promptly can be used to downstream analysis.Parent material is used with Tn5 at 50 ℃ of 5 minutes (Nextera DNA Sample Preparation Kit of LMW; EPICENTRE; GA09115), then 37 degree with S1 nuclease (Promega M5761) is hatched 30 minutes. the dna fragmentation that adds a tail that obtains; With Klenow from 3 ' to the 5 ' circumscribed (NEB that interrupts; 0212L), fast link test kit link joint was used in 20minutes.16 ℃ of 37 ℃ of hatching in 5 minutes. the tagged DNA that obtains carries out pcr amplification (Phusion Hot Start Flex 2X Master Mix, NEB M0536L) according to the PCR operating process of standard with following system. all samples in the process of at every turn handling with enzyme with ZymoDNA Clean and Concentrator kit (Zymo Research Corporation; Irvine; CA) come purifying and enrichment, the PCR product that obtains 1% agarose gel electrophoresis and with SYBR green I Nucleic Acid dyeing (Life technologies, S7567); The picture 3 that resulting result takes with Safe Imager Blue Light Transilluminator (Life technologies, G6600).
Embodiment 3
Adopt the genomic dna of hammer vibrios and with the hammer vibrios DNA that interrupts at round-robin PCR double chain DNA fragment as material, utilize transposase and transposon end sequence (TE) that target DNA is interrupted at random, make it reach the clip size that we expect.The buffer system that material adopts be ((1 * GPS Buffer, 25mMTris-HCl), under different incubation conditions (25 ℃; 2 hours) handle sample, add reaction terminating liquid (10% sucrose, 66mM EDTA then; 20mM TRIS, 0.1%SDS, 0.9%Orange G; And 100g/ml Proteinase K), 10 minutes afterreactions of 50 ℃ of heating can stop.Can under 1% gel electrophoresis, observe cracked dna fragmentation size.
In case the swivel base reaction is accomplished, the dna fragmentation of transposon end sequence (TE) mark is that (Zymo Research Corporation, Irvine CA) carries out purifying to available ZymoDNAClean and Concentrator kit.
(S1nuclease Promega) is hatched at 37 ℃ with the mixture of excision enzyme, up to the terminal equating of the dna fragmentation of strand for the dna fragmentation of transposon end sequence (TE) mark that purifying is good and single stranded DNA restriction endonuclease.Come purifying not have the DNA of mark with ZymoDNA Clean and Concentrator kit.The end sequence of transposon so far is removed, and has produced unlabelled dna fragmentation.
In the unlabelled dna fragmentation of above generation, add the big fragment of dna polymerase i of no 5 prime excision enzyme activity, promptly the Klenow fragment was hatched 20 minutes at 37 ℃.Dna fragmentation through this processing can link to each other with oligonucleotide joint.
The dna fragmentation that does not have mark, barcode label, the restriction enzyme site mark, the mixture of the T4DNA ligase enzyme that the order-checking label of transcripting promoter sequence label and ligation are used was handled 5 minutes or was spent the night at 16 ℃.Add EDTA or stopped ligation in 20 minutes 72 ℃ of reactions.The dna fragmentation library that has label promptly can be used to downstream analysis.
Parent material with Tn5 at 50 ℃ of 5 minutes (Nextera DNA Sample Preparation Kit of LMW; EPICENTRE; GA09115); Then 37 degree with S1 nuclease (Promega M5761) is hatched 30 minutes. the dna fragmentation that adds a tail that obtains with Klenow from 3 ' to 5 ' circumscribed interrupting (NEB, 0212L); 20minutes.16 ℃ of 5 minutes use fast link test kit link order-checking street corner of 37 ℃ of hatchings. the tagged DNA that obtains carries out pcr amplification (Phusion Hot Start Flex 2X Master Mix according to the PCR operating process of standard with following system; NEB M0536L). (Zymo Research Corporation, Irvine CA) come purifying and enrichment to all samples with ZymoDNA Clean and Concentrator kit in the process of at every turn handling with enzyme; The PCR product that obtains is at 1% agarose gel electrophoresis and with SYBRgreen I Nucleic Acid dyeing (Life technologies; S7567), resulting result see the picture 4 that takes with Safe Imager Blue Light Transilluminator (Life technologies, G6600).
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The present invention is described on some details, and the variation of various forms and details can not depart from the true scope of invention.All numerals above-mentioned, form, appendix, patent, patented claim and document give at present and include reference in.
Claims (10)
1. the random fracture and the method for labeled dna fragment simultaneously is characterized in that, may further comprise the steps:
I. transposase, double-stranded transposon and target dna react after mixing, and are exactly under the katalysis of transposase, and double-stranded transposon end sequence inserts in the target dna sequence at random; Formation is by the dna fragmentation of transposon end sequence mark; These fragments from 5 ' to 3 ' start from double-stranded transposon end sequence, are the single stranded DNA sequence of 9 base pairs then; Be double-stranded target dna fragment then, the single stranded DNA sequence of 9 base pairs and double-stranded transposon are terminal;
Ii. remove the double-stranded transposon end sequence that is marked at the DNA two ends, generate unlabelled dna fragmentation.
Iii. the DNA label that connection needs on unlabelled dna segment produces the dna fragmentation that new label is arranged.
2. the method for claim 1 is characterized in that, removes the double-stranded transposon end sequence that is marked at the DNA two ends with the single stranded DNA restriction endonuclease.
3. the method for claim 1 is characterized in that, produces the flat terminal double-stranded DNA of band with the single stranded DNA excision enzyme.
4. the method for claim 1 is characterized in that, it is terminal to produce attachable DNA with archaeal dna polymerase.
5. method as claimed in claim 4 is characterized in that, archaeal dna polymerase is big segment dna polymerase i, T7DNA polysaccharase or T4DNA polysaccharase, or Taq archaeal dna polymerase or do not have the Klennow segment of 3 ' to 5 ' 5 prime excision enzyme activity.
6. the method for claim 1 is characterized in that, unlabelled dna segment is connected the label of needs to be realized with dna ligase.
7. method as claimed in claim 6 is characterized in that, dna ligase is the T4 dna ligase, T7 dna ligase, or Taq archaeal dna polymerase.
8. the method for claim 1 is characterized in that, said DNA label is for being selected from the order-checking label, the barcode label, and the restriction enzyme site mark is caught mark, certification mark, amplification label, or in the transcripting promoter sequence label one or more.
9. method as claimed in claim 8 is characterized in that, the use that can link together of a plurality of labels.
10. random fracture that is used for of using claim 1-9 method, and form the test kit of markd dna fragmentation, comprising the transposon of purifying, the terminal and single stranded DNA nucleicacidase of transposon, archaeal dna polymerase and dna ligase and required label.
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